This is the US National Stagk Application of PCT/FR96/02009 filed Dec. 16, 1996.
The invention relates to novel membrane protein-vinyl polymer of amphiphilic nature water-soluble complexes, to a process for preparing these complexes and to the application of these complexes to diagnostic or analytical methods.
Integral membrane proteins, which are a specific class of proteins, are inserted in vivo into biological membranes and cross the lipid bilayer thereof. The surface of these proteins which naturally comes into contact with the membranes (transmembrane region) is particularly hydrophobic.
Membrane proteins ensure essential biological functions, in particular as regards the exchange of information or of molecules between the various cell compartments and between the cell and its environment. In this respect, they are of major value in the medical field. They represent, for example, preferred targets for medicinal molecules. They are also involved in many human diseases, some of which (for example multiple sclerosis or myasthenia gravis) have an autoimmune component manifested by the presence in the serum of autoantibodies directed against membrane proteins. Manipulation of membrane proteins in aqueous solution is usually a prerequisite which is essential to their purification and to their structural and functional study. It requires avoidance of the spontaneous aggregation of the hydrophobic domains and maintenance of a relatively non-polar environment around the transmembrane regions.
The standard preparations of such proteins in the water-soluble state contain micellar concentrations of surfactants. The success of the process is based on the high affinity of the transmembrane protein regions for these amphiphilic and dispersing compounds. Nevertheless, this is a manipulation which is more intricate than that in the case of soluble proteins, specifically on account of the presence of surfactants.
These surfactants must be added at a concentration above their critical micelle concentration (cmc) to all the solutions containing the test protein. In addition to any problems of cost posed by the consumption of surfactants, the experiments are often made difficult due to the fact that the membrane proteins are usually fragile and sensitive to their environment. For example, in the presence of an excess of micelles, they can become denatured, while a surfactant defect generally leads to their precipitation.
Several patents, among which mention may be made of WO-A-9,400,557; WO-A-9,115,505; EP-A-363 106; DE-A-3 527 139; JP-A-6,107,6500; U.S. Pat. No. 5,223,411; JP-A-0,227,0856 and JP-A-0,116,8653 describe the extraction, purification and manipulation of membrane proteins in aqueous medium. These proteins are either dispersed in micellar systems or are inserted into lipid bilayers.
Schafmeister et al. Science, 262, pp. 734-738, 1993 have also described the formation of complexes between membrane proteins and amphiphilic peptide polymers. The amphiphilic polymers concerned are small polypeptides known as peptitergents, which have rigid structures (xcex1-helices), one face of which is hydrophobic and the other face hydrophilic. Peptitergents maintain the solubility of bacteriorhodopsin. However, they are unsuccessful in the case of a porin, no doubt because their rigidity limits their possibilities of adaptation when faced with various hydrophobic surfaces. The authors envisage the use of peptitergents to facilitate the crystallization of membrane proteins.
Mention will also be made, in the field of combinations between amphiphilic synthetic polymers and globular (water-soluble) proteins, of the studies by F. Petit et al. Sci., 273, pp. 777-781, 1995 on modified amphiphilic polyacrylates with a molecular weight of between 150,000 and 200,000. The aim of these investigations relates to the study of protein/polymer combinations (formation of gels, kinetics and energetics of the complexation, in particular) rather than to the maintenance of membrane proteins as disperse solutions.
There is thus a problem to be solved as regards the manipulation of membrane proteins in detergent-free aqueous solutions in the form of dissolved membrane proteins.
The object of the present invention is to solve this problem in general, and the invention in particular proposes novel membrane protein-vinyl polymer of amphiphilic nature complexes which have the following advantages:
production of concentrated solutions of membrane proteins in native form,
production of freeze-dried preparations of membrane proteins in native form,
low production cost.
The complexes are manipulated in the absence of amphiphilic additives in the medium, which results in a reduction in the purification costs (large volumes of solutions for chromatography, dialyses, etc.).
The term xe2x80x9cvinylxe2x80x9d includes acrylic polymers in its general meaning.
The invention thus relates to a membrane protein-vinyl polymer of amphiphilic nature water-soluble complex, characterized in that said vinyl polymer corresponds to the formula: 
in which:
R1 is a group:
COOxe2x88x92 M+, M+ being an alkali metal cation,
COOR7, R7 being a sugar residue; polyoxyalkylene, in particular polyoxyethylene containing 4 to 10 alkylene oxide units, a radical (CH2)txe2x80x94NR10R11, t being an integer from 1 to 5, R10 and R11, which may be identical or different, being a hydrogen atom or a (C1-C4) alkyl radical,
N-pyrrolidonyl,
phenyl sulfonate,
CONR8R9, R8 and R9, which may be identical or different, being a hydrogen atom, a sugar residue, polyoxyalkylene, in particular polyoxyethylene containing from 4 to 10 alkylene oxide units, or a zwitterionic radical,
R4, R5 and R6, which may be identical or different, are a hydrogen atom or a methyl radical,
R2 is a radical COOR12 or CONR13R14 
R12 being a linear or branched alkyl or alkenyl radical of 6 to 12 carbon atoms,
R13 and R14, which may be identical or different, have one of the meanings of R12, and in addition one of the two can correspond to a hydrogen atom,
R3 is a radical COOR15 or CONR16R17 
R15 being a (C1-C5)alkyl radical,
R16 and R17 having one of the meanings of R15 and in addition it being possible for one of the two to correspond to a hydrogen atom,
x, y and z correspond to the respective percentages of the units,
x being between 20 and 90%
y being between 10 and 80%
z being between 0 and 60%,
the average molar mass being between 500 and 100,000, advantageously less than or equal to 50,000, preferably between 1000 and 50,000.
The average molar mass is given by weight.
The polymers which can be used in the context of the present invention are thus amphiphilic polymers which comprise at least one fatty chain, i.e. a hydrophobic portion and hydrophilic units, i.e. a hydrophilic portion.
The meaning of the various substituents indicated in formula I is given in detail below, as a non-limiting guide:
M+ is a lithium, sodium or potassium alkali metal cation,
R7 is chosen in particular from:
glucose, fructose, maltose and sucrose residues and in general mono- or disaccharide residues; Hxe2x80x94(OCH2xe2x80x94CH2xe2x80x94)4.8; xe2x80x94(CH2)xe2x80x94Nxe2x80x94(CH2xe2x80x94CH3)2 
R8 and R9 are chosen in particular from glucosamine, fructosamine, maltosamine and saccharosamine residues and in general amino mono- or disaccharide residues;
H"Parenopenst"OCH2xe2x80x94CH2"Parenclosest"4.8; 
Among the R12 to R14 alkyl radicals, mention is made in particular of n-hexyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl and n-dodecyl radicals, and C6-C12 radicals containing a secondary carbon or a tertiary carbon.
Among the R12 to R14 alkenyl radicals, mention is made in particular of the linear C6-C12 radicals mentioned above containing one or two double bonds or the same radicals containing a secondary carbon or a tertiary carbon.
Among the R15 to R17 alkyl radicals, mention is made in particular of ethyl, isopropyl, n-propyl, n-butyl, isobutyl, sec-butyl, t-butyl, n-pentyl, sec-pentyl, t-pentyl and isopentyl.
Preferably, the invention relates to membrane protein-acrylic polymer of amphiphilic nature water-soluble complexes, characterized in that said acrylic polymers correspond to the formula: 
M+, R4, R5, R6, R13, R14, R16, R17, x, y and z having the same meaning as in formula I.
The acrylic or vinyl polymers of amphiphilic nature of formula I or II are obtained, in a known manner, from precursor acrylic or vinyl polymers, which may be commercially available or may be synthesized by polymerization of vinyl, acrylic or methacrylic monomers or a mixture of these monomers. In the latter case, copolymers are obtained which, by extension, are included in the general term xe2x80x9cpolymerxe2x80x9d. The acrylic polymers of amphiphilic nature of formula II result from the reaction of compounds R13R14NH and optionally R16R17NH with an acrylic polymer, which leads to a random distribution of the amides throughout the chain. The polymer is placed in salified form beforehand or in a subsequent step. Such a mode of synthesis is described, for example, in March, J. (1985) Advanced Organic Chemistry: Reactions, Mechanisms and Structure, pp. 372-374 (Wiley, New York); Wang, T. K., Iliopoulos, I. and Audebert, R. (1988) Polym. Bull 20, 577-582.